Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins

Niklas Krupka; Padraig Strappe; Jürgen Götz; Lars M. Ittner

doi:10.1016/j.plasmid.2010.01.002

Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins

Niklas Krupka, Padraig Strappe, Jürgen Götz, Lars M. Ittner^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

9 Citations (Scopus)

Abstract

Lentiviral gene delivery has become widely used. Similarly, the Gateway cloning technology that allows restriction-independent cloning of genes into target vectors is becoming increasingly popular. Here, we have generated two Gateway-compatible lentiviral transfer vectors for expression of carboxy-terminal fluorescence tagged fusion proteins, pLVU/GFP and pLVU/RED. We used a restriction enzyme-independent PCR-based approach to introduce the carboxy-terminal fluorescence tags, EmGFP and DsRed, respectively. Both vectors combine the advantages of restriction enzyme/ligation-independent cloning using the Gateway system with a attR1-CmR-ccdB-attR2 recombination cassette, together with expression of fluorescence tagged fusion proteins driven by the robust mammalian ubiquitin C (UbC) promoter. We tested the vectors by expressing different proteins together with the carboxy-terminal fluorescence tags in 293T and SH-SY5Y cells. Both pLVU/GFP and pLVU/RED can be utilized in different experiments, including protein localization studies and live-cell in vivo imaging.

Original language	English
Pages (from-to)	155-160
Number of pages	6
Journal	Plasmid
Volume	63
Issue number	3
DOIs	https://doi.org/10.1016/j.plasmid.2010.01.002
Publication status	Published - 1 May 2010
Externally published	Yes

Keywords

DsRed
Gateway cloning
GFP
Lentiviral vector
Ligation-independent cloning

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10.1016/j.plasmid.2010.01.002

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title = "Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins",

abstract = "Lentiviral gene delivery has become widely used. Similarly, the Gateway cloning technology that allows restriction-independent cloning of genes into target vectors is becoming increasingly popular. Here, we have generated two Gateway-compatible lentiviral transfer vectors for expression of carboxy-terminal fluorescence tagged fusion proteins, pLVU/GFP and pLVU/RED. We used a restriction enzyme-independent PCR-based approach to introduce the carboxy-terminal fluorescence tags, EmGFP and DsRed, respectively. Both vectors combine the advantages of restriction enzyme/ligation-independent cloning using the Gateway system with a attR1-CmR-ccdB-attR2 recombination cassette, together with expression of fluorescence tagged fusion proteins driven by the robust mammalian ubiquitin C (UbC) promoter. We tested the vectors by expressing different proteins together with the carboxy-terminal fluorescence tags in 293T and SH-SY5Y cells. Both pLVU/GFP and pLVU/RED can be utilized in different experiments, including protein localization studies and live-cell in vivo imaging.",

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year = "2010",

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AU - Krupka, Niklas

AU - Strappe, Padraig

AU - Götz, Jürgen

AU - Ittner, Lars M.

PY - 2010/5/1

Y1 - 2010/5/1

N2 - Lentiviral gene delivery has become widely used. Similarly, the Gateway cloning technology that allows restriction-independent cloning of genes into target vectors is becoming increasingly popular. Here, we have generated two Gateway-compatible lentiviral transfer vectors for expression of carboxy-terminal fluorescence tagged fusion proteins, pLVU/GFP and pLVU/RED. We used a restriction enzyme-independent PCR-based approach to introduce the carboxy-terminal fluorescence tags, EmGFP and DsRed, respectively. Both vectors combine the advantages of restriction enzyme/ligation-independent cloning using the Gateway system with a attR1-CmR-ccdB-attR2 recombination cassette, together with expression of fluorescence tagged fusion proteins driven by the robust mammalian ubiquitin C (UbC) promoter. We tested the vectors by expressing different proteins together with the carboxy-terminal fluorescence tags in 293T and SH-SY5Y cells. Both pLVU/GFP and pLVU/RED can be utilized in different experiments, including protein localization studies and live-cell in vivo imaging.

AB - Lentiviral gene delivery has become widely used. Similarly, the Gateway cloning technology that allows restriction-independent cloning of genes into target vectors is becoming increasingly popular. Here, we have generated two Gateway-compatible lentiviral transfer vectors for expression of carboxy-terminal fluorescence tagged fusion proteins, pLVU/GFP and pLVU/RED. We used a restriction enzyme-independent PCR-based approach to introduce the carboxy-terminal fluorescence tags, EmGFP and DsRed, respectively. Both vectors combine the advantages of restriction enzyme/ligation-independent cloning using the Gateway system with a attR1-CmR-ccdB-attR2 recombination cassette, together with expression of fluorescence tagged fusion proteins driven by the robust mammalian ubiquitin C (UbC) promoter. We tested the vectors by expressing different proteins together with the carboxy-terminal fluorescence tags in 293T and SH-SY5Y cells. Both pLVU/GFP and pLVU/RED can be utilized in different experiments, including protein localization studies and live-cell in vivo imaging.

KW - DsRed

KW - Gateway cloning

KW - GFP

KW - Lentiviral vector

KW - Ligation-independent cloning

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JO - Plasmid

JF - Plasmid

SN - 0147-619X

IS - 3

ER -

Gateway-compatible lentiviral transfer vectors for ubiquitin promoter driven expression of fluorescent fusion proteins

Abstract

Keywords

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